Detection of the phosphatase activity of carbonic anhydrase III on a nitrocellulose membrane following 2D gel electrophoresis

Abstract

Carbonic anhydrase isozyme III (CAIII) is unique among the carbonic anhydrases because it exhibits phosphatase activity. CAIII is relatively specific to skeletal muscles, and may therefore be a useful diagnostic marker for muscular diseases. In the muscles of patients with myasthenia gravis (MG), CAIII is deficient and previous studies have demonstrated that changes in the phosphatase activity of CAIII is a fundamental mechanism underlying the weakness and fatigability of MG. However, there have been no effective analytical methods for investigating its phosphatase activity until now. In the present study, a new method combining two-dimensional electrophoresis (2-DE) and phosphatase staining in situ on a nitrocellulose membrane was reported to detect the phosphatase of CAIII in skeletal muscle extracts. Furthermore, a recombinant CAIII was constructed and its phosphatase activity staining was demonstrated to be positive. This method allows for the effective detection of the phosphatase activity of CAIII following 2-DE and is a promising technique for functional proteomics.